Project Summary/Abstract The PI requests support of his research program in Iron Trafficking and Regulation in Biological Systems. Using powerful biophysical, bioanalytical, and mathematical tools, the PI and his coworkers propose to characterize and quantify the major iron species in isolated organelles, whole cells and organs from a wide diversity of biological systems. Mssbauer spectroscopy will be the primary biophysical tool, with EPR and UV-vis spectroscopies playing supporting roles. Various genetic strains of budding yeast and human cells will be enriched in 57Fe. Mitochondria, cytosol, vacuoles and other organelles will be isolated. The major Fe species in these cellular components will be probed. Ordinary-differential-equations-based mathematical models will be developed to analyze the results obtained and to generate a system?s level description of Fe trafficking and regulation. No other group worldwide investigates Fe in this way. Yeast strains are being investigated in which Mrs3/4, high-affinity Fe importers located on the mitochondrial inner membrane, have been deleted and overexpressed. Other Fe-associated yeast strains, as well as human Jurkat cells in which mitoferrins (homologs of Mrs3/4) and ferritin are knocked- down, will also be investigated. The Fe content of Escherichia coli and other prokaryotes will be similarly explored. The central bioanalytical tool will be a liquid chromatography system located in a refrigerated anaerobic glove box that is linked to an on-line inductively coupled plasma mass spectrometer AND to an on-line electrospray ionization mass spectrometer. This LC- (ICP)-ESI-MS system will be unique worldwide. It will be used to characterize dozens of labile low-molecular-mass (LMM) metal complexes that the PI and his group have discovered in organelles, cytosol, blood plasma, and in E. coli. A LMM Fe complex in mitochondria that has a mass of ca. 580 Da (called Fe580) is actively under investigation. Fe580 is most probably used as feedstock for iron-sulfur cluster (ISC) assembly and for the iron-insertion step of heme biosynthesis. The composition of a LMM sulfur-containing species called X-S will also be investigated. X-S is exported from mitochondria in association with ISC assembly, and it may be used to assemble cytosolic ISCs and to regulate Fe trafficking. A LMM Fe species in blood plasma called ?non-transferrin-bound iron? or NTBI will be investigated using pigs into which a feeding tube and sampling catheters have been surgically implanted. NTBI damages organs in patients with Fe-overload diseases such as hemochromatosis. Various strains of mice with Fe- associated diseases will be investigated to determine the form of Fe that accumulates in their organs. The effect of hypoxia on Fe accumulation will also be examined.